(i) Field of the Invention
The present invention relates to a method for the preparation of DNA virus vectors capable of replicating in eukaryotic as well as in prokaryotic cells, and to the DNA virus vectors prepared by said method as well as to cells containing said vectors.
(ii) Description of Related Art
Epstein-Barr virus is one of several herpes viruses in humans. The DNA sequence of the EBV B95.8 isolate has been determined (Baer et al., 1984), and detailed scientific evidence has been worked out mainly with respect to DNA elements which play important roles in the two EBV phases. In the so-called `latent phase` the virus establishes a stable host cell relation during which the vitality of the cell remains unaffected, however, the viral DNA genome is replicated in the form of an extrachromosomal plasmid in the host cells and passed on into the daughter cells. The latent phase may be associated with a transformation or immortalization, respectively, of the cells infected in a latent manner. The replication origin of the plasmid, oriP, is the DNA element essential for maintenance and replication of the EBV genome in the latent phase. (Yates et al., 1985). This DNA element is also active in recombinant plasmids and has been used as such in several ways.
In the so-called `lytic or productive phase` the virus is produced in active manner which in addition to the expression of almost all of the viral proteins necessary for regulation of expression or for expression of structural viral components involves two other DNA elements of the virus: the lytic origin of replication, oriLyt, is responsible for viral DNA amplification (Hammerschmidt and Sugden, 1988), the terminal repeats, TR, are packaging signals indispensable for encapsidation of the amplified EBV DNA (Hammerschmidt and Sugden, 1989).
There is a broad interest in the genetic analysis of EBV functions as well as the construction of recombinant EBV genomes. The interest is based on the fact that EBV genomes may bear additional therapeutical genes or that certain undesired properties or genes of EBV may be removed. The techniques used so far in the alteration of the EBV genome are based on homologous recombination events of recombinant E. coli plasmids with EBV genomes in cell lines infected by EBV in the latent manner (Cohen et al., 1989; Hammerschmidt and Sugden, 1989). Alternatively, so-called mini EBV genomes may be prepared in E. coli which, however, only contain the viral gene functions important for the latent phase of EBV (Sugden and Hammerschmidt, 1993; Hammerschmidt and Sugden, 1995; Kempkes et al., 1995a; Kempkes et al., 1995b).